This invention relates to variants of herpes simplex virus type 1 (HSV-1) which lack neurovirulence. Such variants are of value in the preparation of live attenuated vaccines for the prevention of HSV infections in humans.
Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are important human pathogens which infect more than 80% of the general population and cause recurrent mucocutaneous lesions. Following replication HSV enters the peripheral nervous system where active replication is turned off by an unknown mechanism. Thereafter a latent infection in neurons is established which persists for the life of the host. HSV can reactivate from the latent state to produce infectious lesions. HSV is responsible for a broad spectrum of clinical diseases ranging from relatively benign cutaneous lesions to fatal viral encephalitis.
A considerable amount of research has already been devoted to elucidation of the genetic organisation of both HSV-1 and HSV-2. The HSV-1 genome is a linear double stranded DNA molecule of approximately 152 kilobase pairs consisting of two components L and S. Each component consists of unique sequences U1 and Us, flanked by inverted repeats. The organisation of the HSV-2 genome is similar but not identical. For a detailed description of the genetic organisation of HSV-1 and HSV-2 (see McGeoch, 1987).
The identification of genes involved in viral pathogenicity and the elucidation of their precise functions is of fundamental importance to the understanding of the biology of herpes simplex virus (HSV). A number of variants of both HSV type 1 (strain 17) and HSV type 2 (strain HG52) with defined deletions in the unique and repeat sequences of both the long and short regions of the viral genome have already been isolated and characterised (Brown et al 1984, Harland and Brown 1985, Brown and Harland 1987, MacLean and Brown, 1987a and b, Harland and Brown 1989). Little is known, however, about the molecular mechanisms which regulate the neurovirulence of HSV. It has been shown that a deletion variant of HSV-2 strain HG52, termed JG2604, is avirulent on intracerebral inoculation of mice (Taha et al, 1989a). JH2604 has xe2x80x94 1488 base pair deletion within both copies of the long repeat region of the genome [i.e. terminal long repeat (TRL) and internal inverted long repeat (IRL) regions].
An HSV-1 strain 17/HSV-2 strain HG52 recombinant (initially isolated in the Institute of Virology, Glasgow by Marsden et al, 1978), termed RE6, has also been reported to be avirulent in mice (Thompson et al 1989).
In HSV-1, inverted repeats of the L component designated ab and bxe2x80x2axe2x80x2 are each approximately 9 Kbp whereas those of the S component cxe2x80x2axe2x80x2 and ca, are each approximately 6.5 Kbp. A sequence shared by the inverted repeats of the L and S components is designed the xe2x80x98axe2x80x99 sequence. This sequence has been reported (Chou and Roizman 1986) to contain the promoter-regulatory sequence and the transcription initiation sites for a diploid gene located in the b sequence of the inverted repeats of the L component. Working with HSV strain F these authors reported that there was a transcribed open reading frame (ORF) between the xe2x80x98axe2x80x99 sequence and an immediate early gene designated IE1. By the use of antipeptide sera they were able to show that the ORF specified a protein designated ICP 34.5 (Ackermann et al 1986). Recently Chou and Roizman (1990) have reported that their now predicted ORF is conserved in 2 other HSV-1 strains analysed but not in Glasgow strain HSV-1 (17) syn+. It has been suggested by Chou et al (1990) that the neurovirulence locus of HSV-1 comaps with, and requires the expression of, ICP 34.5.
Surprisingly it has now been found that HSV-1 Glasgow strain 17 variants modified in the terminal portion of RL lack neurovirulence.
Such variants are incapable of replicating in CNS neurons, but are able, in mice to elicit a good immunological and cell mediated response since they are capable of replication in the peripheral tissue. This ability emphasises the vaccine potential of these strains.
According to the present invention there is provided an HSV-1 strain the genome of which is modified in the terminal portion of RL within Bam HI s (0-0.02 and 0.81-0.83 mu).
By Bam HI s it will be appreciated that what is meant is each copy of the approximately 3 Kb Bam HI s fragment of the HSV RL region.
The term xe2x80x98modifiedxe2x80x99 is used herein to denote disruption of the Bam HI s fragment by deletion of one or more nucleotides, insertion of additional nucleotides or any other alteration of the nucleotide sequence such as rearrangement, or substitution.
The HSV-1 strain may be a spontaneously isolated deletion variant of may be a wild type strain into which the desired modification has been introduced.
Such modifications in the HSV strain may be made by genetic manipulation, for example by site-directed mutagenesis, or by excision of a portion of the genome with or without replacement with a pre-prepared DNA cassette incorporating the required modification. Alternatively one may isolate naturally occurring HSV-1 variants, e.g. deletion variants.
Preferably the HSV-1 strain of the invention is a Glasgow strain 17 variant.
In one preferred aspect the HSV-1 strain is a strain in which at least 100 nucleotides in the Bam HI sxe2x80x2 region between the Alu I site at 125074 np and 125972 np within the a sequence and its counterpart sequence in TRL have been deleted.
More preferably 0.5 to 3 kb of the Bam HI sxe2x80x2 region and its counterpart in TRL is deleted, still more preferably about 0.7-2.5 kb is deleted.
In one specific example the HSV-1 variant is a strain designated 1714 which is a spontaneously occurring deletion variant of variant 1702 and lacks 759 bp within each copy of the Bam HI s fragment of the RL region as described hereinbelow, in which the deletion associated with non-neurovirulence is located between nucleotide positions 125213 and 125972.
Such a deletion removes one complete copy of the 18 bp DR1 element of the xe2x80x98axe2x80x99 sequence and terminates 1105 bp upstream of the 5xe2x80x2 end of the immediate early gene 1.
In another specific example the HSV-1 variant is a variant designated 1716 in which the 759 bp deletion in variant 1714 has been introduced into the wild type Glasgow strain 17+.
In order to understand the invention more clearly reference may be made to FIG. 1 hereinbelow in which:
(a) Shows the HSV-1 genome (with map units marked) in the prototype orientation; and
(b) Shows an expansion of BamHI k(s+g). The BamHI (B) and AluI (A) sites flanking the deletion in 1714/1716 are marked. All coordinates are based on the numbering of McGeoch et al (1988). Also indicated are the positions of the 5xe2x80x2 end of IE1, the xe2x80x98axe2x80x99 sequence, the DR1/Ub boundary in the xe2x80x98axe2x80x99 sequence, a 189 bp conserved open reading frame between HSV-1 and HSV-2 (RL ORF) and the end points of the 759 bp deletion in 1714/1716. The deletion extends from the DR1/Ub boundary to remove the 5xe2x80x2 107 bp of the RL ORF.
The present invention further provides a whole virus vaccine comprising an HSV-1 strain according to the invention wherein such vaccine comprises an immunoprotective and non-toxic amount of the strain of the invention. Such vaccine may comprise the strain alone or in conjunction with other antigens and/or adjuvants.
Due to their non-pathogenic nature, the viruses of the present invention are exceptional candidates for further modification. For example they may be further modified so as to carry heterologous antigens. The virus can be engineered so as to express antigens from HSV-2, such as HSV-2 gD. Such a virus, elicits both antibody and CTL responses to both type 1 and type 2 virus and, moreover, enhances the overall immune response. Similarly other antigens from the other pathogens may be presented by the viruses of the present invention. For example, gene products from HCMV, VZV, EBV, HHV6, HHV7, and HIV as well as other envelope viruses may be presented.
Moreover, the virus of the present invention may be modified by introducing a mutation, typically a temperature sensitive mutation into the gene UL26a which encodes the capsid protein, P40 (Liu and Roizman 1991 a+b).
Such a mutation at non-permissive temperatures, (typically 38.5xc2x0 C.) results in the overproduction of light particles; that is virus particles lacking the nucleocapsid and nucleic acid, and hence infectivity. J. of Gen Virology (1991) 72 p661 Szilagyi and Cunningham.
Accordingly the present invention provides for light particles derived from the viruses described herein.
In a further embodiment, the present invention provides herpetic virus light particles carrying a heterologous antigen. For example in one embodiment of the present invention HSV-1 1716 has been modified to express HSV-2 gD, and also modified to contain a temperature sensitive mutation in UL26a gene at 38.5xc2x0 C.; this mutant over produces light particles containing HSV-2 gD. Other HSV-2 protein maybe incorporated into such a virus, in particular the HSV-2 gene products ICPO, ICP4 and Vmw 65 kD. Membrane proteins from other herpetic virus such as HCMV, VZV, EBV, HHV6, HHV7, and other enveloped virus such HIV-1 and HIV-2 maybe presented. For example gB from HCMV, gp120 from HIV-1 or HIV2 maybe incorporated into the virus Light particle. In theory any heterologous membrane protein which does not interfere with viral entry into the cell, can be carried by the light particles according to the invention.
Accordingly, the present invention provides a herpetic viral light particle carrying a heterologous antigen. In particular, the present invention provides a herpes simplex virus, preferably type 1, light particle carrying a heterologous antigen. An embodiment of this aspect of invention is HSV-1 1716, gD1+, gD2+, UL26a ts and light particles derived therefrom.
The Light particles of the present invention may be prepared by a modification of the method of Szilagyi and Cunningham (supra). Briefly cells are infected at 5 pfu/cell at the non-permissive temperature (npt) 38.5xc2x0 C. and the supernatant virus harvested at 30 hours post infection. This preparation is centrifuged on a preformed 5-15% Ficoll (made in Eagle""s medium) gradient for 2 hours at 12 K. The Light particle band is removed with a 26 G needle and pelleted at 20 K overnight in normal cell growth medium (Eagles).
The light particles of the present invention are useful for vaccine purposes. Accordingly in a further aspect of the present invention there is provided a vaccine comprising a light particle from a herpetic virus carrying a heterologous antigen. In a further aspect there is provided a vaccine comprising an HSV-1 viral light particle derived from a virus comprising a modification in the terminal portion of RL within BamH1 s (0-0.02 and 0.81-0.83 mu).
Alternatively, or in addition to the above mentioned modification(s), a virus of the present invention may be modified by introducing a mutation, typically a deletion, which renders the LAT promoter ineffective. Such a mutation adds a further level of safety, reducing both the frequency and rate of reactivation from latency.
Accordingly the present invention provides an HSV-1 virus modified in the terminal portion of RL within BamHI s (0-0.02 and 0.81-0.83 mu) and also modified to render the LAT promoter ineffective. Such a modified virus may be further modified so as to produce heterologous antigens such example HSV-2 gD, in the manner contemplated above. Moreover, additionally or alternatively to expressing a heterologous antigen, a temperature sensitive mutation maybe incorporated into the gene UL26a, so as to enable the overproduction of light particles and thus reduce the amount of potentially infective virus present. Such light particles may be separated from infective virus by Ficoll centrifugation of a viral particle. Normally, the ratio of heavy to Light particles in the Light particle band would be 1:103, however where a mutation in UL26a has been incorporated, the ratio of heavy to Light particles is typically in the order of 1:106.
The invention also provides a process for preparing a whole virus vaccine, which process comprises admixing the strain according to the invention with a suitable carrier or adjuvant.
For the preparation of a live attenuated vaccine, standard methodology may be used.
In a further aspect, the invention provides a method of treating HSV infection in humans, which method comprises administering to a human subject in need thereof an immunologically effective dose of the vaccine according to the invention.
The mode of administration of the vaccine of the invention may be any suitable route which delivers an immunoprotective amount of the strain or Light particles of the invention to the subject. However, the vaccine is preferably administered parenterally via the intramuscular or deep subcutaneous routes. Other modes of administration may also be employed, where desired, such as oral administration or via other parenteral routes, i.e., intradermally, intranasally, or intravenously.
The appropriate immunoprotective and non-toxic dose of such vaccine can be determined readily by those skilled in the art, i.e., the appropriate immunoprotective and non-toxic amount of the strain or Light particle of this invention contained in the vaccine of this invention may be in the range of the effective amounts of antigen in conventional whole virus vaccines. It will be understood, nowever, that the specific dose level for any particular patient will depend upon a variety of factors including the age, general health, sex, and diet of the patient; the time of administration; the route of administration; synergistic effects with any other drugs being administered; and the degree of protection being sought. Of course, the administration can be repeated at suitable intervals if necessary.
The following examples illustrate the invention.